LOCUS       sequence-224302-        6043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control of EKARet-cyto.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6043)
  AUTHORS   Tang S, Yasuda R
  TITLE     Imaging ERK and PKA Activation in Single Dendritic Spines during 
            Structural Plasticity.
  JOURNAL   Neuron. 2017 Mar 22;93(6):1315-1324.e3. doi: 
            10.1016/j.neuron.2017.02.032. Epub 2017 Mar 9.
  PUBMED    28285819
REFERENCE   2  (bases 1 to 6043)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2017.02.032"; journalName: "Neuron"; date: 
            "2017-03-22- 22"; volume: "93"; issue: "6"; pages: "1315-1324.e3"
COMMENT     Sequence Label: negEKARet-cyto
FEATURES             Location/Qualifiers
     source          1..6043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1058..1060,1061..1063,1064..1774)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITLGMDELYK"
     CDS             1949..2296
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             join(2363..2365,2366..2368,2369..3079)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2363..2365,2366..2368,2369..3079)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    3158..3279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3169..3188)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3223..3242
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3460..3915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3547..3566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3757..3778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3982..4001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4101..4123
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4161..4179)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4247..4351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4352..4420,4421..5212)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4570..4589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5383..5970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5871..5890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggcg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcatg gtgagcaagg gcgaggagct
     1081 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1141 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctcat
     1201 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
     1261 cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1321 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1381 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1441 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actggaacag
     1501 cgtcaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1561 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1621 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
     1681 gttcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcc tgaccgccgc
     1741 cgggatcact ctcggcatgg acgagctgta caagctcgag atggcggacg aggagaagct
     1801 gccgcccggc tgggagaagc gcatgagccg cagctcaggc cgagtgtact acttcaacca
     1861 catcactaac gccagccagt gggagcggcc cagcggcaac agcagcagtg gtggcaaaaa
     1921 cgggcagggg gagcctgcca ggggtaccag tgctggtggt agtgctggtg gtagtgctgg
     1981 tggtagtgct ggtggtagtg ctggtggttc cggcagtgct ggtggtagtg ctggtggtag
     2041 taccagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     2101 tggttccggc agtgctggtg gtagtgctgg tggtagtacc agtgctggtg gtagtgctgg
     2161 tggtagtgct ggtggtagtg ctggtggtag tgctggtggt tccggcagtg ctggtggtag
     2221 tgctggtggt agtaccagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     2281 tggtagtgct ggtggttccg gaccagatgt ccctagagct ccagtggata aagcaaagct
     2341 gtcattccaa tttccgacgc gtatggtgag caagggcgag gagctgttca ccggggtggt
     2401 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2461 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2521 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2581 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2641 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2701 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2761 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2821 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2881 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2941 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3001 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3061 catggacgag ctgtacaagg atgctcttca aaaaaaactt gaagaacttg aacttgacga
     3121 atgaactagt atctgcggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     3181 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3241 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3301 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     3361 aaatgtggta aaatcgataa ggatccgggc tggcgtaata gcgaagaggc ccgcaccgat
     3421 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat
     3481 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3541 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3601 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3661 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3721 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3781 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3841 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3901 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3961 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4021 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4081 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4141 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4201 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4261 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4321 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4381 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4441 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4501 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4561 taaagtgctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4621 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4681 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4741 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4801 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4861 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4921 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4981 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5041 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5101 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5161 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5221 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5281 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5341 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5401 gcgcgtaatc tgctgcttgc aaacaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5461 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5521 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5581 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5641 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5701 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5761 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5821 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5881 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5941 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6001 ctggcctttt gctggccttt tgctcacatg gctcgacaga tct
//
